The field of the present invention is the area of DNA repair enzymes. In particular, the invention concerns the identification of stable ultraviolet DNA endonuclease polypeptide fragments, their nucleotide sequences and recombinant host cells and methods for producing them and for using them in DNA repair processes.
Cellular exposure to ultraviolet radiation (UV) results in numerous detrimental effects including cell death, mutation and neoplastic transformation. Studies indicate that some of these deleterious effects are due to the formation of two major classes of bipyrimidine DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4 PPs). (Friedberg et al. [1995] in DNA Repair and Mutagenesis, pp. 24-31, Am. Soc. Microbiol., Washington, D.C.).
Organisms have evolved several different pathways for removing CPDs and 6-4 PPs from cellular DNA (Friedberg et al. [1995] supra; Brash et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 8810124-10128). These pathways include direct reversal and various excision repair pathways which can be highly specific or nonspecific for CPDs and 6-4 PPs. For example, DNA photolyases specific for either CPDs or 6-4 PPs have been found in a variety of species and restore the photoproduct bases back to their original undamaged states (Rubert, C. S. [1975] Basic Life Sci. 5A:73-87; Kim et al. [1994] J. Biol. Chem. 269:8535-8540; Sancar, G. B. [1990] Mutat. Res. 236:147-160). Excision repair has been traditionally divided into either base excision repair (BER) or nucleotide excision repair (NER) pathways, which are mediated by separate sets of proteins but which both are comprised of DNA incision, lesion removal, gap-filling and ligation reactions (Sancar, A. [1994] Science 266:1954-19560; Sancar, A. and Tang, M. S. [1993] Photochem. Photobiol. 57:905-921). BER N-glycosylase/AP lyases specific for CPDs cleave the N-glycosidic bond of the CPD 5xe2x80x2 pyrimidine and then cleave the phosphodiester backbone at the abasic site via a xcex2-lyase mechanism, and have been found in several species including T4 phage-infected Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae (Nakabeppu, Y. et al. [1982] J. Biol. Chem. 257:2556-2562; Grafstrom, R. H. et al. [1982] J. Biol. Chem. 257:13465-13474; Hamilton, K. K. et al. [1992] Nature 356:725-728). NER is a widely distributed, lesion non-specific repair pathway which orchestrates DNA damage removal via a dual incision reaction upstream and downstream from the damage site, releasing an oligonucleotide containing the damage and subsequent gap filling and ligation reactions (Sancar and Tang [1993] supra).
Recently, an alternative excision repair pathway initiated by a direct acting nuclease which recognizes and cleaves DNA containing CPDs or 6-4 PPs immediately 5xe2x80x2 to the photoproduct site has been described (Bowman, K. K. et al. [1994] Nucleic. Acids Res. 22:3026-3032; Freyer, G. A. et al. [1995] Mol. Cell. Biol. 15:4572-4577; Doetsch, P. W. [1995] Trends Biochem. Sci. 20:384-386; Davey, S. et al. [1997] Nucleic Acids Res. 25:1002-1008; Yajima, H. et al. [1995] EMBO J. 14:2393-2399; Yonemasu, R. et al. [1997] Nucleic Acids Res. 25:1553-1558; Takao, M. et al. [1996] Nucleic Acids Res. 24:1267-1271). The initiating enzyme has been termed UV damage endonuclease (UVDE, now termed Uve1p). Homologs of UVDE have been found in Schizosaccharomyces pombe, Neurospora crassa and Bacillus subtilis (Yajima et al. [1995] supra; Yonemasu et al. [1997] supra; Takao et al. [1996] supra). The Uve1p homologs from these three species have been cloned, sequenced and confer increased UV resistance when introduced into UV-sensitive strains of E. coli, S. cerevisiae, and human cells (Yajima et al. [1995] supra; Takao et al, [1996] supra). In S. pombe Uve1p is encoded by the uve1+ gene. However, because of the apparently unstable nature of partially purified full-length and some truncated UVDE derivatives, UVDE enzymes have been relatively poorly characterized and are of limited use (Takao et al. [1996] supra).
Because of the increasing and widespread incidence of skin cancers throughout the world and due to the reported inherent instability of various types of partially purified full-length and truncated UVDE derivatives, there is a long felt need for the isolation and purification of stable UVDE products, especially for use in skin care and medicinal formulations.
It is an object of the present invention to provide purified stable UVDE (Uve1p), polypeptide fragments which retain high levels of activity, particularly those from the Schizosaccharomyces pombe enzyme. In a specific embodiment, the polypeptide fragment is xcex94228-UVDE, which contains a 228 amino-acid deletion of the N-terminal region of the S. pombe uve1+ gene product; a second specific embodiment is the fusion protein GST-xcex94228-UVDE. The DNA sequence encoding GST-full-length UVDE from S. pombe is given in SEQ ID NO:1. The deduced amino acid sequence of full-length GST-UVDE is given in SEQ ID NO:2. The DNA sequence encoding xcex94228-UVDE is given in SEQ ID NO:3. The deduced amino acid sequence of xcex94228-UVDE is given in SEQ ID NO:4. The DNA coding sequence and deduced amino acid sequence for GST-xcex94228-UVDE are given in SEQ ID NO:5 and SEQ ID NO: 6, respectively. Also encompassed within the present invention are truncated UVDE proteins wherein the truncation is from about position 100 to about position 250 with reference to SEQ ID NO:2, and wherein the truncated proteins are stable in substantially pure form.
Also within the scope of the present invention are nucleic acid molecules encoding such polypeptide fragments and recombinant cells, tissues and animals containing such nucleic acids or polypeptide fragments, antibodies to the polypeptide fragments, assays utilizing the polypeptide fragments, pharmaceutical and/or cosmetic preparations containing the polypeptide fragments and methods relating to all of the foregoing.
A specifically exemplified embodiment of the invention is an isolated, enriched, or purified nucleic acid molecule encoding xcex94228-UVDE. Another exemplified embodiment is an isolated, enriched or purified nucleic acid molecule encoding GST-xcex94228-UVDE.
In a specifically exemplified embodiment, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5.
In another embodiment, the invention encompasses a recombinant cell containing a nucleic acid molecule encoding xcex94228-UVDE or GST-xcex94228-UVDE. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, a synonymous coding sequence or a functional derivative of SEQ ID NO:3 or SEQ ID NO:5. In such cells, the xcex94228-UVDE coding sequence is generally expressed under the control of heterologous regulatory elements including a heterologous promoter that is not normally coupled transcriptionally to the coding sequence for the UVDE polypeptide in its native state.
In yet another aspect, the invention relates to a nucleic acid vector comprising a nucleotide sequence encoding xcex94228-UVDE or GST-xcex94228-UVDE and transcription and translation control sequences effective to initiate transcription and subsequent protein synthesis in a host cell. Where a GST full length or truncated derivative is expressed, the GST portion is desirably removed (after affinity purification) by protease cleavage, for example using thrombin.
It is yet another aspect of the invention to provide a method for isolating, enriching or purifying the polypeptide termed xcex94228-UVDE.
In yet another aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a UVDE polypeptide fragment. By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to UVDE polypeptides with greater affinity than it binds to other polypeptides under specified conditions.
Antibodies having specific binding affinity to a UVDE polypeptide fragment may be used in methods for detecting the presence and/or amount of a truncated UVDE polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the UVDE polypeptide. Kits for performing such methods may be constructed to include a first container having a conjugate of a binding partner of the antibody and a label, for example, a radioisotope or other means of detection as well known to the art.
Another embodiment of the invention features a hybridoma which produces an antibody having specific binding affinity to a UVDE polypeptide fragment. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody, for example a xcex94228-UVDE specific antibody. In preferred embodiments, the UVDE specific antibody comprises a sequence of amino acids that is able to specifically bind xcex94228-UVDE. Alternatively, a GST-tag specific antibody or labeled ligand could be used to determine the presence of or quantitate a GST-xcex94228-UVDE polypeptide, especially in formulations ex vivo.
The present invention further provides methods for cleaving DNA molecules at positions with structural distortions, wherein the DNA is cleaved in the vicinity of the distortion by a stable truncated UVDE protein of the present invention. The structural distortion can result from mismatch at the site of the distortion in a double-stranded DNA molecule, from UV damage or from other damage to DNA due to chemical reaction, for example, with an alkylating or depurination agent or due to damage due to UV irradiation, ionizing radiation or other irradiation damage. The stable truncated UVDE proteins can be supplied in substantially pure form for in vitro reactions or they can be supplied for in vivo reactions, including but not limited to compositions for topical application (in the form or of an ointment, salve, cream, lotion, liquid or transdermal patch) in pharmaceutical compositions for internal use (to be administered by intraperitoneal, intradermal, subcutaneous, intravenous or intramuscular injection). The stable truncated UVDE derivatives of the present invention repair a wide variety of mismatch and DNA damage. The cleavage of a double stranded DNA molecule having structural distortion due to nucleotide mispairing (mismatch) or due to DNA damage by a stable truncated UVDE derivative of the present invention can be used to advantage in a relatively simple assay for structural distortion wherein cleavage of a test molecule (i.e., the double stranded DNA molecule which is being screened for damage, mismatch or other structural distortion) is to be detected.
The present invention further provides a method for cleaning a double stranded DNA molecule in which there is a structural distortion. The structural distortion can be due to aberrations including, but not limited to, base pair mismatch, photoproduct formation, alkylation of a nucleotide such that normal Watson-Crick base pairing is disturbed, intercalation between nucleotides of a compound which could be, for example, an acriflavine, an ethidium halide, among others, or a platinum adduct, for example of a cisplatin moiety. The DNA can also contain an abasic site, an inosine, xanthine, 8-oxoguanine residue, among others. The method of the present invention can be employed using the UVDE (Uve1p) protein from Schizosaccharomyces pombe, a truncated derivative of the S. pombe UVDE (lacking from about 100 to about 250 N-terminal amino acids), the xcex94228-UVDE of S. pombe, or the Neurospora crassa, Bacillus subtilis, Homo sapiens or Deinococcus radiodurans enzymes as set forth herein (see SEQ ID NOs.:36-39). A specifically exemplified truncated UVDE (xcex94228) is given in SEQ ID NO:4. DNA containing the structural distortion is contacted with an enzyme (or active truncated derivative) as described above under conditions allowing endonucleolytic cleavage of one strand of the distorted DNA molecule.